**4.1 Biological basis for therapeutic applications: EVs as mediators of intercellular interactions**

EVs are secreted by most cell types and are taken up by recipient cells, where their cargo consisting of a cocktail of proteins, mRNAs and non-coding RNAs alters the behavior of the recipient cells in a way that might be even considered similar to hormones or cytokines [74], e.g. in the context of skin or bone cell paracrine signaling [75–77]. siRNAs (small interfering RNAs) and miRNA-based inhibitors have been recognized as potent novel drug candidates for many years. As EVs can be loaded with different drugs *in vitro*, they qualify as an attractive drug delivery system. The specificity of the recipient cell targeting *in vivo* is understood in a limited way only, although there is evidence of accumulation of specific EVs [43]. For example, EVs from human mesenchymal stem cells accumulated in the liver, spleen and sites of acute kidney injury [78]. Such tropism for a specific cell type, a requirement for targeted drug delivery, appears to be determined by surface proteins of the source cells. The composition of EV membrane reflecting the one of their source cell makes these particles non-immunogenic, and their small size allows them to pass the immune surveillance of the host organism [79, 80]. The reported engagement of exosomes in physiological processes in normal and diseased central nervous system makes them attractive vehicles for delivering neurotherapeutic agents across the blood-brain barrier [81–83]. Nevertheless, their delivery in humans seems so far limited to liver and kidney as they are reported not to reach therapeutic amounts in brain, heart and other tissues due to lack of specific targeting and thus low enrichment of the intended therapeutic ingredient in the target tissue. Modifications of the EV surface membrane to achieve enhanced targeting of a specific cell type are hence a common strategy embodied in several different engineering approaches.

*Current Topics in Biochemical Engineering*

membrane components.

expression of CD69 [58].

to facilitate raft association [9].

**3.4 Tetraspanins in pathogen infection**

**3.3 The role of tetraspanins in immune complexes**

cellular ectodomain cleavage and release activity of these enzymes [46]. The tetraspanins of the TspanC8 group (tetraspanins with 8 cysteines) have a significant impact on the cellular exit and catalytic activity of ADAM10 [47], in particular the activity of Tspan15/ADAM10 promoted N-cadherin cleavage [48–50]. Different TspanC8/ ADAM10 complexes seem to have different substrate specificities [51]. The silencing

Important discovery of the possible consequences of fine differences in composition of TEMs has been delivered by the study of exosomes enriched in Tspan8-α<sup>4</sup> complex that were preferentially taken up by the endothelial and pancreatic cells [53]. The fact that such modifications can allow selective targeting *in vitro* and *in vivo* holds promise to achieve improved exosomal delivery by engineering of their

In antigen-presenting cells (APCs), tetraspanins integrate into TEMs proteinrecognition receptors binding to conserved repeated motifs of microbes, such as Toll-like receptors, and MHCII molecules into tetraspanin web platforms, as well as Fcγ receptor I in phagocytic cells, Fcγ receptor IIb and III upon the activation of macrophages and Fcε receptor I in monocytes and skin-derived dendritic cells [54]. The particular role of CD81 protein in the formation of specialized microdomains in the plasma membrane of the cells of the immune system was discovered by elucidating its function of recruiting various adhesion molecules, receptors and signaling proteins to the central zone of the immune synapse in T-lymphocytes and APCs [55]. Therefore, it has early been proposed for CD81 to play a key role during antigenic presentation, since it colocalizes with the T-cell receptor/CD3 [56], and CD81 indeed turned out to be a regulator of CD3 clustering and sustained CD3 signaling [57]. Further, the T-cell side of the immune synapse is densely populated by tetraspanins CD9 and CD151. The abolishment of their expression reduces markers of activation of T-lymphocytes conjugated to the APCs, such as IL-2 secretion and

Another role in the immune response of tetraspanin CD81 is amplifying and sustaining B-cell receptor signaling from lipid rafts by ligation to the co-receptor CD19/CD21 complex. The signaling through a variety of cell surface protein complexes implies a role of lipid rafts, again highlighting the ability of tetraspanin

Several studies have been oriented towards the research of tetraspanins as ligand molecules for pathogen entry. CD81 has been identified as a ligand for hepatitis C virus (HCV) recognition and viral entry [59]. The ligand for viral glycoprotein E2 is the D-domain of the LEL, a dynamic region positioned within the triple-bundle helix, whose conformation in solution differs substantially from the one suggested by crystal structure. Challenging for structure-based design, this region nevertheless presents an attractive target for design of therapeutically relevant ligands with methods such as NMR [60]. Apart of the EC2 domain, other regions of CD81 have proven important for virus infection. Experimental evidence here was based on the exchange of the structural domains of the molecule with the ones of tetraspanins of different degrees of homology, and it was found that closely related substitutions were more efficient at functional complementation of CD81. Viral entry has been shown to correlate with surface expression of the chimeric protein and to depend on the presence of the cholesterol-coordinating glutamate residue [61]. EWI-2wint,

of CD9 enhanced shedding of ADAM17-substrates TNF-α and ICAM-1 [52].

**110**
